FLOURY ENDOSPERM11-2 encodes plastid HSP70-2 involved with the temperature-dependent chalkiness of rice (Oryza sativa L.) grains.

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein 2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

Targeted exome sequencing of unselected heavy-ion beam-irradiated populations reveals less-biased mutation characteristics in the rice genome.

Heavy-ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost-efficient whole-exome sequencing procedure in rice, and its application to characterize an unselected population of heavy-ion beam-induced mutations. The bioinformatics pipeline identified single-nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy-ion beams at the population level. In total, 165 individual M2 lines derived from six irradiation conditions as well as eight pools from non-irradiated 'Nipponbare' controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon-ion beam-induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M2 lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing-based determination of the optimal irradiation condition for induction of mutations.

Heavy-ion beam mutagenesis identified an essential gene for chloroplast development under cold stress conditions during both early growth and tillering stages in rice.

We isolated a cold sensitive virescent1 (csv1) mutant from a rice (Oryza sativa L.) population mutagenized by carbon ion irradiation. The mutant exhibited chlorotic leaves during the early growth stages, and produced normal green leaves as it grew. The growth of csv1 plants displayed sensitivity to low temperatures. In addition, the mutant plants that were transferred to low temperatures at the fifth leaf stage produced chlorotic leaves subsequently. Genetic and molecular analyses revealed translocation of a 13-kb genomic fragment that disrupted the causative gene (CSV1; LOC_Os05g34040). CSV1 encodes a plastid-targeted oxidoreductase-like protein conserved among land plants, green algae, and cyanobacteria. Furthermore, CSV1 transcripts were more abundant in immature than in mature leaves, and they did not markedly increase or decrease with temperature. Taken together, our results indicate that CSV1 supports chloroplast development under cold stress conditions, in both the early growth and tillering stages in rice.

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll - protein complexes during leaf senescence.

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll - protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4-1, a stay-green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4-1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation-related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity-dependent manner. Surprisingly, the Fv /Fm value remained high in nyc4-1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4-1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild-type and other non-functional stay-green mutants, including sgr-2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll - protein complexes during leaf senescence is discussed.

A green-cotyledon/stay-green mutant exemplifies the ancient whole-genome duplications in soybean.

The recent whole-genome sequencing of soybean (Glycine max) revealed that soybean experienced whole-genome duplications 59 million and 13 million years ago, and it has an octoploid-like genome in spite of its diploid nature. We analyzed a natural green-cotyledon mutant line, Tenshin-daiseitou. The physiological analysis revealed that Tenshin-daiseitou shows a non-functional stay-green phenotype in senescent leaves, which is similar to that of the mutant of Mendel's green-cotyledon gene I, the ortholog of SGR in pea. The identification of gene mutations and genetic segregation analysis suggested that defects in GmSGR1 and GmSGR2 were responsible for the green-cotyledon/stay-green phenotype of Tenshin-daiseitou, which was confirmed by RNA interference (RNAi) transgenic soybean experiments using GmSGR genes. The characterized green-cotyledon double mutant d1d2 was found to have the same mutations, suggesting that GmSGR1 and GmSGR2 are D1 and D2. Among the examined d1d2 strains, the d1d2 strain K144a showed a lower Chl a/b ratio in mature seeds than other strains but not in senescent leaves, suggesting a seed-specific genetic factor of the Chl composition in K144a. Analysis of the soybean genome sequence revealed four genomic regions with microsynteny to the Arabidopsis SGR1 region, which included the GmSGR1 and GmSGR2 regions. The other two regions contained GmSGR3a/GmSGR3b and GmSGR4, respectively, which might be pseudogenes or genes with a function that is unrelated to Chl degradation during seed maturation and leaf senescence. These GmSGR genes were thought to be produced by the two whole-genome duplications, and they provide a good example of such whole-genome duplication events in the evolution of the soybean genome.

Defect in non-yellow coloring 3, an alpha/beta hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice.

Chlorophyll degradation is an important phenomenon in the senescence process. It is necessary for the degradation of certain chlorophyll-protein complexes and thylakoid membranes during leaf senescence. Mutants retaining greenness during leaf senescence are known as 'stay-green' mutants. Non-functional type stay-green mutants, which possess defects in chlorophyll degradation, retain greenness but not leaf functionality during senescence. Here, we report a new stay-green mutant in rice, nyc3. nyc3 retained a higher chlorophyll a and chlorophyll b content than the wild-type but showed a decrease in other senescence parameters during dark incubation, suggesting that it is a non-functional stay-green mutant. In addition, a small amount of pheophytin a, a chlorophyll a-derivative without Mg(2+) ions in its tetrapyrrole ring, accumulated in the senescent leaves of nyc3. nyc3 shows a similar but weaker phenotype to stay green (sgr), another non-functional stay-green mutant in rice. The chlorophyll content of nyc3 sgr double mutants at the late stage of leaf senescence was also similar to that of sgr. Linkage analysis revealed that NYC3 is located near the centromere region of chromosome 6. Map-based cloning of genes near the centromere is very difficult because of the low recombination rate; however, we overcame this problem by using ionizing radiation-induced mutant alleles harboring deletions of hundreds of kilobases. Thus, it was revealed that NYC3 encodes a plastid-localizing alpha/beta hydrolase-fold family protein with an esterase/lipase motif. The possible function of NYC3 in the regulation of chlorophyll degradation is discussed.

Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice.

Yellowing, which is related to the degradation of chlorophyll and chlorophyll-protein complexes, is a notable phenomenon during leaf senescence. NON-YELLOW COLORING 1 (NYC1) in rice encodes a membrane-localized short-chain dehydrogenase/reductase (SDR) that is thought to represent a chlorophyll b reductase necessary for catalyzing the first step of chlorophyll b degradation. Analysis of the nyc1 mutant, which shows the stay-green phenotype, revealed that chlorophyll b degradation is required for the degradation of light-harvesting complex II and thylakoid grana in leaf senescence. Phylogenetic analysis further revealed the existence of NYC1-LIKE (NOL) as the most closely related protein to NYC1. In the present paper, the nol mutant in rice was also found to show a stay-green phenotype very similar to that of the nyc1 mutant, i.e. the degradation of chlorophyll b was severely inhibited and light-harvesting complex II was selectively retained during senescence, resulting in the retention of thylakoid grana even at a late stage of senescence. The nyc1 nol double mutant did not show prominent enhancement of inhibition of chlorophyll degradation. NOL was localized on the stromal side of the thylakoid membrane despite the lack of a transmembrane domain. Immunoprecipitation analysis revealed that NOL and NYC1 interact physically in vitro. These observations suggest that NOL and NYC1 are co-localized in the thylakoid membrane and act in the form of a complex as a chlorophyll b reductase in rice.

Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice.

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a mechanism distinct from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10-7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

A novel carotenoid derivative, lutein 3-acetate, accumulates in senescent leaves of rice.

The biosynthetic pathway of lutein, the most abundant carotenoid in leaves, has been extensively studied, while its degradation pathway during senescence is poorly understood. We found that a novel carotenoid derivative, lutein 3-acetate, accumulates in senescent leaves of rice (Oryza sativa L.). The change in contents of lutein and lutein 3-acetate suggests that lutein is converted to lutein 3-acetate during senescence. Analysis of mutants involved in light-harvesting complex II (LHCII) accumulation and degradation suggests that the converting activity is induced during senescence and that only free lutein, not lutein bound to LHCII, is converted into lutein 3-acetate.

Development of PCR markers to detect the glb1 and Lgc1 mutations for the production of low easy-to-digest protein rice varieties.

Limiting the ingestion of protein is the fundamental idea in the diet therapy for patients with chronic renal failure. Two mutations involved in the content of major rice storage proteins useful for developing low easy-to-digest protein rice variety have been isolated. The glb1 mutation causes the deficiency of alpha-globulin, and the Lgc1 mutation reduces the glutelin content. By combining the glb1 and the Lgc1 mutations, it is possible to reduce the easy-to-digest protein content by approximately 50%. The Lgc1 mutation has been shown to be caused by a 3.5-kb deletion between the glutelin structural genes, GluB4 and GluB5, while the molecular basis of glb1 mutation has been less understood. PCR analysis of the glb1 mutation revealed a 62.8-kb deletion, including the structural gene of alpha-globulin. Based on these lines of information, we generated PCR markers that make it possible to detect the glb1 and Lgc1 mutations. Using those PCR markers, we genotyped F(2) plants segregating for the glb1 mutation and the Lgc1 mutation and confirmed the consistency of genotype and phenotype. Because the PCR marker sets can distinguish heterozygotes, they will be very useful in developing new varieties of low easy-to-digest protein rice.

Molecular characterization of mutations induced by gamma irradiation in rice.

In order to analyze mutations induced by gamma irradiation in higher plants, we irradiated rice with gamma rays and screened for mutations expressing phenotypes of glutinous endosperm (wx), chlorophyll b deficiency, endosperm protein deficiency, gibberellin-related dwarfism, and shortened plastochron-in order to clarify types of mutations. Nucleotide sequence analysis showed that the most frequent mutation induced by gamma rays was deletion, particularly small deletion. Of the 24 mutations, 15 were small deletions (1-16 bp), four were large deletions (9.4-129.7 kbp), three were single-base substitutions, and two were inversions. Deletions 100 bp-8 kbp in length were not found, suggesting that gamma irradiation is unlikely to induce deletions of 100 bp to 8 kbp but is more likely to induce deletions between 1 and several ten bp or those of around 10 kbp or more. Based on the results, reverse genetics applications may be possible for gamma irradiation-induced deletions in rice by mismatch cleavage analysis used in Targeting Induced Local Lesions IN Genomes (TILLING) to detect small deletions and base substitutions or by using array comparative genomic hybridization (aCGH) to detect large deletions.

LET dependence on killing effect and mutagenicity in the model filamentous fungus Neurospora crassa.

PURPOSE: To assess the unique biological effects of different forms of ionizing radiation causing DNA double-strand breaks (DSBs), we compared the killing effect, mutagenesis frequency, and mutation type spectrum using the model filamentous fungus Neurospora. MATERIALS AND METHODS: Asexual spores of wild-type Neurospora and two DSB repair-deficient strains [one homologous recombination- and the other non-homologous end-joining (NHEJ) pathway-deficient] were irradiated with argon (Ar)-ion beams, ferrous (Fe)-ion beams, or X-rays. Relative biological effectiveness (RBE), forward mutation frequencies at the ad-3 loci, and mutation spectra at the ad-3B gene were determined. RESULTS: The canonical NHEJ (cNHEJ)-deficient strain showed resistance to higher X-ray doses, while other strains showed dose-dependent sensitivity. In contrast, the killing effects of Ar-ion and Fe-ion beam irradiation were dose-dependent in all strains tested. The rank order of RBE was Ar-ion > Fe-ion > C-ion. Deletion mutations were the most common, but deletion size incremented with the increasing value of linear energy transfer (LET). CONCLUSIONS: We found marked differences in killing effect of a cNHEJ-deficient mutant between X-ray and high-LET ion beam irradiations (Ar and Fe). The mutation spectra also differed between irradiation types. These differences may be due to the physical properties of each radiation and the repair mechanism of induced damage in Neurospora crassa. These results may guide the choice of irradiation beam to kill or mutagenize fungi for agricultural applications or further research.

Knockout of glutelin genes which form a tandem array with a high level of homology in rice by gamma irradiation.

In the course of evolution, a gene is often duplicated in tandem, resulting in a functional redundancy. The analysis of function of these genes by raising double mutant might be difficult because they are very tightly linked. We described here a mutant of such a tandem duplicated gene. glu1 is a gamma-ray-induced rice mutant, which lacks an acidic subunit of glutelin, a major seed storage protein. We found that glu1 harbors a 129.7-kb deletion involving two highly similar and tandem repeated glutelin genes, GluB5 and GluB4. The deletion eliminated the entire GluB5 and GluB4 gene except half of the first exon of GluB5. GluB5 and GluB4 have the same amino acid sequence in the acidic subunit, suggesting that only the mutation involving both GluB5 and GluB4 results in the lack of the glutelin acidic subunit deleted in glu1. Our finding suggests that gamma-ray can be an effective mutagen to analyze tandem repeated and functionally redundant genes.

Linear Energy Transfer-Dependent Change in Rice Gene Expression Profile after Heavy-Ion Beam Irradiation.

A heavy-ion beam has been recognized as an effective mutagen for plant breeding and applied to the many kinds of crops including rice. In contrast with X-ray or γ-ray, the heavy-ion beam is characterized by a high linear energy transfer (LET). LET is an important factor affecting several aspects of the irradiation effect, e.g. cell survival and mutation frequency, making the heavy-ion beam an effective mutagen. To study the mechanisms behind LET-dependent effects, expression profiling was performed after heavy-ion beam irradiation of imbibed rice seeds. Array-based experiments at three time points (0.5, 1, 2 h after the irradiation) revealed that the number of up- or down-regulated genes was highest 2 h after irradiation. Array-based experiments with four different LETs at 2 h after irradiation identified LET-independent regulated genes that were up/down-regulated regardless of the value of LET; LET-dependent regulated genes, whose expression level increased with the rise of LET value, were also identified. Gene ontology (GO) analysis of LET-independent up-regulated genes showed that some GO terms were commonly enriched, both 2 hours and 3 weeks after irradiation. GO terms enriched in LET-dependent regulated genes implied that some factor regulates genes that have kinase activity or DNA-binding activity in cooperation with the ATM gene. Of the LET-dependent up-regulated genes, OsPARP3 and OsPCNA were identified, which are involved in DNA repair pathways. This indicates that the Ku-independent alternative non-homologous end-joining pathway may contribute to repairing complex DNA legions induced by high-LET irradiation. These findings may clarify various LET-dependent responses in rice.

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway.

Mutants that retain greenness of leaves during senescence are known as "stay-green" mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl-protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes.

Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence.

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)-bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.

Assessment of utility of meiosis-associated promoters of lily for induction of germinal ds transposition in transgenic rice.

In order to suppress the somatic excision of the Ds element and increase the independent transposition events of the Ac/Ds transposon tagging system in rice, we employed promoters of two meiosis-specific genes of lily, LIM10 and LIM18. The LIM10 promoter directed GUS expression specifically in anthers, with the LIM18 promoter doing the same in the anthers and somatic tissue. Both promoters induced independent germinal transposition with the frequency of approximately 1%. The LIM10 promoter, lacking induction of somatic transposition, is considered to be useful for improving transposon-tagging efficiencies in rice.